Decreased fertility in later reproductive age in women is an increasing trend due to career-building, as ages of first marriage and first birth increase. In addition, acute cancer treatment and chronic disease also lead to loss or decrease in fertility. In order to preserve fertility, advances in our understanding of oocyte and embryo health are required during cryo-preservation and thawing. An understanding of the health of the oocyte or embryo depends on knowledge of the stress during cryopreservation, handling, thawing, and reintroduction into culture. AMPK regulates oocyte maturation during stress and regulates a transcription factor maintaining potency in 2-cell embryos, blastocysts, and blastocyst-derived placental trophoblast stem cells (TSC). AMPK is primarily an energy sensor and suppresses anabolic processes and enhances catabolic processes to regain ATP homeostasis. In an arctic frog species, AMPK is induced during freeze and thaw to levels similar to those our laboratory has observed in stressed embryos and TSC. We hypothesize that AMPK is the key enzyme regulating homeostatic and developmental processes during oocyte freeze-down and thaw. Thus, we also hypothesize that measuring and managing the levels of AMPK activation will facilitate the optimization of oocyte cryopreservation and culture. To test this hypothesis, we will measure AMPK activity in oocytes during pre-freeze handling, after freeze and after thaw and match these levels with those of age matched oocytes in vivo. We will also add doses of AMPK agonist or antagonist during prefreeze handling, freeze down medium, and during culture after thaw, IVF and culture to blastocyst stage. Several landmarks of IVF and embryo development during culture will be measured to determine efficacy of AMPK agonists and antagonists in optimizing cryopreservation. This proposal is innovative in managing AMPK that is emerging as the primary enzyme coregulating homeostatic and developmental responses to stress and known to be regulated during freeze and thaw in arctic frogs. PUBLIC HEALTH RELEVANCE: Every year, nearly 300,000 American women of reproductive age undergo cancer treatment or experience chronic diseases that threaten fertility. These women need a robust technique for preserving fertility, and oocyte cryopreservation will fulfill this need if it can be made more reliable. AMPK activity is important in normal and stressed oocyte maturation. AMPK is important in freezing and thawing of an arctic frog species suggesting it is part of a natural process regulating responses to freeze and thaw. We hypothesize that AMPK is the key enzyme regulating homeostatic and developmental processes during oocyte freeze-down and thaw and will measure AMPK activity during cryopreservation and manage AMPK activity to optimize oocyte cryopreservation.